Ca Sparks and Secretion in Dorsal Root Ganglion Neurons

Ca2؉ sparks and secretion in dorsal root ganglion neurons

Kunfu Ouyang*†‡, Hui Zheng*‡§, Xiaomei Qin¶, Chen Zhang*§, Dongmei Yang*†, Xian Wang¶, Caihong Wu*†, Zhuan Zhou*†§ʈ, and Heping Cheng*ʈ**

*Institute of Molecular Medicine and †National Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing, China; §Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; ¶Department of Physiology, Health Science Center, Peking University, Beijing 100083, China; and **Laboratory of Cardiovascular Sciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892

Edited by Ramon Latorre, Center for Scientific Studies, Valdivia, Chile, and approved July 15, 2005 (received for review January 6, 2005) Ca2؉ sparks as the elementary intracellular Ca2؉ release events are sensing and nociceptive inputs to the spinal cord and higher central .(instrumental to local control of Ca2؉ signaling in many types of nervous system (CNS -cells. Here, we visualized neural Ca2؉ sparks in dorsal root ganglion To trigger exocytosis, local Ca2ϩ must exceed several (in chro (DRG) sensory neurons and investigated possible role of DRG maffin cells) or 10 micromolar (in terminals) (15), and such high sparks in the regulation of secretion from the somata of the cell. concentrations can only be achieved in the vicinity of a Ca2ϩ DRG sparks arose mainly from type 3 ryanodine receptor Ca2؉ channel. Intriguingly, in many vertebrate and invertebrate neurons release channels on subsurface cisternae of the endoplasmic retic- of both the CNS and peripheral systems, including DRG, special- ulum, rendering a striking subsurface localization. Caffeine- or ized regions of the ER, dubbed subsurface cisternae (SSC), come 3,7-dimethyl-1-(2-propynyl)xanthine-induced store Ca2؉ release, in into direct apposition to the plasma membrane, forming nanoscopic the form of Ca2؉ sparks, triggered exocytosis, independently of junctions reminiscent of the dyads or triads in muscles (16, 17). At membrane depolarization and external Ca2؉. The spark–secretion places of SSC, Ca2ϩ release might occur spontaneously or it can be coupling probability was estimated to be between 1 vesicle per 6.6 coupled to local Ca2ϩ entry via the CICR mechanism and, conse- sparks and 1 vesicle per 11.4 sparks. During excitation, subsurface quentially, elevate local Ca2ϩ concentrations to trigger exocytosis of .sparks were evoked by physiological Ca2؉ entry via the Ca2؉- nearby vesicles induced Ca2؉ release mechanism, and their synergistic interaction In the present study, we investigated local Ca2ϩ signaling between with Ca2؉ influx accounted for Ϸ60% of the Ca2؉-dependent surface membrane and SSC and its role in the regulation of exocytosis. Furthermore, inhibition of Ca2؉-induced Ca2؉ release secretion in DRG sensory neurons. In particular, we demonstrated abolished endotoxin-induced secretion of pain-related neuropep- that Ca2ϩ release from type 3 RyRs on SSC, in the form of ‘‘Ca2ϩ tides. These findings underscore an important role for Ca2؉ sparks sparks’’ (18), can occur spontaneously or provoked by caffeine and in the amplification of surface Ca2؉ influx and regulation of neural its analog 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX). During secretion. excitation, similar sparks are triggered by and amplify the surface Ca2ϩ influx. These subsurface sparks play an important role in endoplasmic reticulum ͉ ryanodine receptor ͉ exocytosis triggering and modulating vesicular secretion from the somata of DRG neurons. a2ϩ is a ubiquitous intracellular messenger that regulates vital Cphysiological processes ranging from synaptic transmission, Methods muscle contraction, hormone secretion to cell survival, and cell Cells and Patch Clamp Recordings. Freshly isolated rat DRGs (C5– Ϫ death (1–3). In neurons, as in other types of cells, the cytoplasmic L5) were prepared with collagenase (1.5 mg⅐ml 1) and tripsin (1 Ϫ Ca2ϩ originates from two sources, Ca2ϩ ingress through voltage- mg⅐ml 1) at 37°C, as described (11). Cells were used 1–10 h after and ligand-gated Ca2ϩ channels in the plasma membrane and Ca2ϩ preparation. Only the small (15–25 ␮m, C-type) neurons without release from internal stores, mainly the endoplasmic reticulum apparent processes were used. Whole-cell patch-clamp recordings (ER) (4). Two types of Ca2ϩ channels, namely ryanodine receptors were performed in either perforated or broken-in configuration by ͞ PULSE (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs), consti- using an EPC9 2 amplifier and software (HEKA Elektronik, tute the pathways for Ca2ϩ release of the ER. The RyR pathway, Lambrecht͞Pfalz, Germany). Standard external solution contained ϩ ϩ which operates via the Ca2 -induced Ca2 release (CICR) mech- 150 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 10 mM anism, is found in a variety of neurons (5–9), and affords the Hepes, and 10 mM glucose, at pH 7.4. The patch pipette (2–4 M⍀) ϩ possibility for excitation-Ca2 release coupling and for generation filling solution contained 150 mM CsCl, 4.0 mM MgCl2,10mM of Ca2ϩ signal independently of excitation (7). Hepes, and 0.15 mM nystatin (omitted in the broken-in configu- The Ca2ϩ-dependent vesicular secretion of transmitters and ration), at pH 7.2. All experiments were performed at room peptides is widely involved in neural, paracrine, and endocrine temperature (22–25°C). functions. Most neurons release their neurotransmitters in its ؉ terminals at the synapse in a Ca2ϩ-dependent manner. However, Ca2 Imaging. DRG neurons were loaded with fluo-4 AM (5 ␮M, Huang and Neher (10) reported that depolarization can trigger 30 min) (Molecular Probes), and imaged with a Zeiss 510 inverted

capacitance increase in small dorsal root ganglion (DRG) neurons PHYSIOLOGY (15–25 ␮m in diameter, C-type), indicating somatic exocytosis. Using amperometry and imaging technique, we have recently This paper was submitted directly (Track II) to the PNAS ofﬁce. Abbreviations: ER, endoplasmic reticulum; RyR, ryanodine receptor; CICR, Ca2ϩ-induced detected quantal release of preloaded transmitters or dyes from the 2ϩ Ca release; IP3R, inositol 1,4,5-trisphosphate receptor; DRG, dorsal root ganglion; CGRP, DRG somata in response to action potentials (11, 12). Among calcitonin gene-related peptide; SSC, subsurface cisternae; DMPX, 3,7-dimethyl-1-(2- others, pain-related peptides such as calcitonin gene-related pep- propynyl)xanthine; Cm, membrane capacitance. tide (CGRP) and substance P may be released from the somata of ‡K.O. and H.Z. contributed equally to this work. the cell (13, 14). Thus, the somatic secretion most likely provides a ʈTo whom correspondence may be addressed. E-mail: [email protected] or way for an excited DRG neuron to modulate the activity of adjacent [email protected]. ganglion neurons (including itself) and thereby the transmission of © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408494102 PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12259–12264 Downloaded by guest on September 24, 2021 confocal microscope with ϫ40 oil immersion lens (numerical aperture 1.3). The horizontal and axial resolutions were 0.4 and 1.5 ␮m, respectively. Line scan, curve scan, and 2D imaging modes were used to measure sparks and Ca2ϩ transients. Extracellular solution contained 150 mM NaCl, 5.0 mM KCl, 1.1 mM MgCl2,10 mM glucose, 10 mM Hepes, and 2.0 mM CaCl2 (pH 7.4). In some experiments, caffeine or DMPX was added to nominal zero Ca2ϩ solution and delivered to cells by locally placed glass pipette. Image processing and data analysis were performed by using IDL 5.4 software (Research Systems, Boulder, CO). Automated detection and analysis of sparks used the computer algorithm developed by Cheng et al. (19). Fitting the spatial and temporal profiles of sparks used the procedure described in refs. 20 and 21.

Membrane Capacitance (Cm) Measurement. The Cm was measured with standard whole-cell patch-clamp configuration by using the software lock-in module of PULSE 8.30 together with an EPC9͞2 amplifier. A 1-kHz, 40-mV peak-to-peak sinusoid was applied around a DC holding potential of Ϫ80 mV. The resulting current was analyzed by using the Lindau–Neher technique to give esti-

mates of the Cm, membrane conductance, and series resistance (22). 2ϩ Patch pipette (2–3 M⍀) filling solution contained 150 mM CsCl, 5.0 Fig. 1. Ca sparks in DRG neurons. (A) Sequential confocal images of a DRG neuron in the presence of 1 mM caffeine. Images were taken 0.25 s apart. (B) mM MgATP, 0.1 mM Li4GTP, 10 mM Hepes, and 0.25 mM fluo-4 Ca2ϩ sparks along the cell diameter (ii) and their inhibition by ryanodine (10 or 0.1 mM fura-2 potassium salt. Voltage commanding pulses were ␮M, 20 min) (iii). (i) Scan line position. (C) Radial distribution of sparks, f(R Ϫ synchronized to confocal imaging by marking the image with a 1-ms r), where R is the radius of the cell and (R Ϫ r) is the distance from surface. The LED flash at the onset of the Cm recording. number of sparks in a shell (r) should be proportional to 4␲r2f(RϪ r), and Ϸ75% of sparks were restricted to the 1-␮m outermost shell. (D) Subsurface RIA of CGRP Release. DRG neurons were enzymatically isolated localization of RyR3 immunofluorescence. No specific immunoreactivity was from neonate rats as described (23). Cells were maintained in observed for anti-RyR1,2 and anti-RyR2 antibodies (Upper). (Lower Right) The culture for Ϸ3–6 days before CGRP release assay. After washing punctuated RyR3 immunofluorescence on cell surface flattened by a coverslip. with DMEM, cultured DRG neurons were incubated in 1 ml of DMEM, to measure the basal release of CGRP, or in DMEM neurons. Furthermore, DRG sparks persisted upon brief removal containing designated reagents. After 20-min incubation, the su- ϩ ϩ of external Ca2 (n ϭ 8 cells), excluding Ca2 influx as the direct pernatants were removed from the culture wells, and the amounts ϩ source of Ca2 . of CGRP-like immunoactivity (LI) were measured with use of CGRP RIA as described (24). Subsurface Localization of DRG Sparks. Mapping the spark-igniting sites uncovered a striking subsurface localization of DRG sparks. Immunolabelling. Dissociated DRG neurons were fixed and incu- Of 238 sparks observed along the diameter of DRG cells, 133 (56%) bated overnight with primary monoclonal antibodies that recognize were found within 1 ␮m of the surface (Fig. 1C). Considering the RyR2, RyR1,2 (both RyR1 and RyR2), and RyR1,3 (both RyR1 spherical geometry of the cell body, we estimated that Ϸ75% sparks and RyR3) (25) at 4°C, then incubated with Cy3-conjugated ␮ anti-mouse antibodies (Jackson ImmunoResearch) for 1.0 h in the are localized to the outmost 1- m shell of the cytoplasm (Fig. 1). dark. To determine the subcellular organization of RyRs participating in the genesis of sparks, we performed immunocytochemical assay Statistics. Data were given as mean Ϯ SEM. The significance of using antibodies reacting with different RyR isoforms. Our data difference between means was determined, when appropriate, by showed the presence of type 3, but neither type 1 nor type 2 RyR using nonparametric Kruskal–Wallis test, Student’s t test, and reactivity in DRG cells (Fig. 1D), in agreement with a recent report paired t test. P Ͻ 0.05 was considered statistically significant. (27). RyR3 immunofluorescence was sharply enriched at punctuated sites, and such ‘‘puncta’’ were seen most abundant along a shell Results immediately beneath the cell surface (Fig. 1D). By focusing on the -Ca2؉ Sparks in DRG Neurons. Small sensory neurons from rat DRGs coverslip-flattened cell surfaces, we estimated the density of sub ␮ 2 were examined with confocal microscopy in conjunction with the surface RyR3 to be 0.33 sites per m (Fig. 1D). Tissue staining of Ca2ϩ indicator, fluo 4, under conditions conducive to CICR (1 mM entire ganglions further revealed that almost all neurons are caffeine to sensitize RyRs). Time-lapsed confocal imaging revealed equipped with such subsurface RyR3 puncta (data not shown). sudden, local, and short-lived elevations of the cytoplasmic Ca2ϩ, It was shown in early 1960s that, in many types of neurons namely Ca2ϩ sparks, at the periphery of the cell body (Fig. 1A). including DRG cells, SSC make contacts with the plasma mem- High-resolution line scan imaging along the cell diameter showed brane at the somata and proximal processes (16), but their physi- that individual sparks remain highly confined in space and time, ological significance remains elusive. In this regard, the aforemen- with no incidence of activating regenerative Ca2ϩ waves (Fig. 1B). tioned functional and immunocytochemical data strongly suggest ϩ DRG sparks were abolished by RyR antagonists, ryanodine at a that SSC represent the Ca2 release units that give rise to sparks in high concentration (10 ␮M for 20 min, n ϭ 15 cells; Fig. 1B), and DRG neurons. These uniquely placed subsurface sparks from tetracaine at 1 mM (n ϭ 6 cells), indicating the RyR origin of sparks. RyR3 on SSC might be instrumental to reciprocal communication 2ϩ However, DRG sparks could not be prevented by the IP3R antag- between the plasma membrane and the intracellular Ca stores. onist xestospongin C (10 ␮M, 10 min, n ϭ 11 cells) or 2-amino- ethoxydiphenyl borate (2-APB, 20 ␮M, 10 min, n ϭ 10 cells). Triggering Sparks by Physiological Ca2؉ Entry. Given the subsurface Although 2-APB at high concentrations (50–100 ␮M) blocks also localization of RyR3 and sparks, it is possible that membrane the store-operated Ca2ϩ entry (26), these data collectively suggest excitation of DRG cells is coupled to spark production, translating 2ϩ that IP3Rs are not required for the genesis of sparks in DRG neural activity into intracellular Ca signal. To investigate the

12260 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408494102 Ouyang et al. Downloaded by guest on September 24, 2021 Fig. 3. Properties of neuronal Ca2ϩ sparks. (A) A typical spark obtained in the presence of 1 mM caffeine. (B–D). Histograms of spark amplitude (B), width (C), and release duration (from the onset to the beginning of decay) (D). Exponential ﬁt (smooth curve) to the data in D, excluding the ﬁrst data point, yielded a time constant (␶) 33.2 ms.

Fig. 2. Depolarization-evoked sparks. (A) Spontaneous sparks in the absence of caffeine. (Inset) Confocal curved line scan (red) at a repetition rate of 330 bution for spark spatial width (Fig. 3C). The release duration of Hz. (B) Small depolarization activated subsurface sparks under the perforated DRG sparks followed a monoexponential distribution (time con- whole-cell patch clamp conditions. Ryanodine (10 ␮M) or thapsigargin (TG, 10 stant ␶ ϭ 33.2 ms; Fig. 3D), as if DRG spark termination is governed ␮ 2ϩ M) or removal of extracellular Ca (Lower) abolished depolarization- by a first-order stochastic process. Notably, many DRG spark- induced sparks. (C) Depolarization to the apparent reversal potential of Ca2ϩ currents (ϩ80 mV) failed to elicit any sparks. igniting sites manifested repetitive activity (Figs. 1 and 2), suggesting that DRG spark production exhibits little use-dependent refractoriness. hypothetical excitation–spark coupling, we used the ‘‘curve scan’’ Regardless whether they were spontaneous events or caffeine- or imaging technique in which the scanning laser beam tracks a curved depolarization-evoked ones, DRG sparks displayed similar ampli- trajectory underneath the cell surface (Fig. 2A Inset). Subsurface tudes (⌬F͞F0 ϭ 0.27 Ϯ 0.05, 0.32 Ϯ 0.01 and 0.33 Ϯ 0.01, n ϭ 13, sparks in the absence of caffeine occurred at a rate of 0.40 Ϯ 0.16 293, and 40 for spontaneous, caffeine-, and depolarization-evoked (100 ␮m)Ϫ1⅐sϪ1 (n ϭ 18 cells; Fig. 2A), whereas 1 mM caffeine events, respectively), spatial spread (full width at half maximum ϭ increased the spark rate to 10.3 Ϯ 0.8 (100 ␮m)Ϫ1⅐sϪ1 (n ϭ 41 cells, 2.03 Ϯ 0.29, 2.05 Ϯ 0.05 and 1.98 Ϯ 0.26 ␮m), and release duration P Ͻ 0.01 vs. control). Fig. 2B Top illustrates that, in a voltage- (37.9 Ϯ 6.3, 45.9 Ϯ 2.6 and 47.5 Ϯ 7.7 ms). The similarities of DRG clamped cell, near-threshold depolarization from holding potential sparks under various experimental conditions support the notion Ϫ70 mV to Ϫ55 mV evoked sparks amidst an elevating Ca2ϩ that sparks constitute the elementary Ca2ϩ release events in DRG background [1.58 Ϯ 0.46 (100 ␮m) Ϫ1⅐sϪ1, n ϭ 15 cells, P Ͻ 0.05 vs. neurons. That caffeine increases the propensity of spark occurrence control]. The depolarization-evoked sparks were indistinguishable without affecting their unitary properties suggests that caffeine from the spontaneous sparks in the absence of caffeine (Fig. 2A), specifically modulates the activation, but not termination, of the and were abolished by ryanodine [0.03 Ϯ 0.03 (100 ␮m) Ϫ1⅐sϪ1, n ϭ elementary release events in DRG neurons. 17, P Ͻ 0.01 vs. untreated cells] or thapsigargin pretreatment [0.04 Ϯ 0.04 (100 ␮m) Ϫ1⅐sϪ1, n ϭ 8, P Ͻ 0.05 vs. untreated cells; Fig. DRG Sparks Directly Trigger Exocytosis. Because the vast majority of 2B]. However, the dim and homogenous Ca2ϩ background was sparks are precisely localized to the membrane subspace (Fig. 1), it ryanodine- and thapsigargin-resistant (Fig. 2B), consistent with the is tempting to speculate that subsurface sparks are involved in 2ϩ notion that it arises from voltage-gated Ca currents (ICa), such as regulating the release of vesicles in their immediate vicinities. Next, 2ϩ those of L- N-, P-, and Q-type high voltage-activated and T-type low we sought to test this possibility by measuring Cm during store Ca 2ϩ voltage-activated Ca channels in the plasma membrane (28, 29). release. Because Cm is proportional to the surface area of the cell, 2ϩ Depolarization in the absence of extracellular Ca (Fig. 2B; n ϭ stimulus-induced capacitance increase (⌬Cm) provides a sensitive 10 cells) or to the apparent reversal potential of ICa (ϩ80 mV, Fig. measurement of vesicle fusion in somata of DRG neurons (10). This 2C; n ϭ 12 cells) failed to elicit any increase in local sparks or fusion is thought to be a key event for delivering the bioactive cargos background Ca2ϩ elevation. These findings indicate that Ca2ϩ entry (e.g., CGRP and substance P) into the extracellular space to affect

via ICa, rather than the membrane depolarization per se, is oblig- possible autocrine and paracrine functions. Caffeine or its analog, PHYSIOLOGY atory to ignition of sparks during excitation. Taken together, we DMPX, at 5 mM in Ca2ϩ-free solution evoked a large number of conclude that DRG sparks are coupled to membrane excitation by discrete sparks that were quickly fused into global Ca2ϩ transients the CICR mechanism. (Fig. 4 A and C). The average transient amplitude were ⌬F͞F0 ϭ 1.33 Ϯ 0.13 (caffeine, n ϭ 8) or 1.43 Ϯ 0.15 (DMPX, n ϭ 13), Unitary Properties of DRG Sparks. The unitary properties of DRG respectively. Assuming that sparks constitute the building blocks of sparks were measured by fitting the spatial profiles, the rise and the global Ca2ϩ transients and based on unitary properties of decay time courses of individual sparks to Gaussian and monoex- sparks, we estimated that an average subsurface Ca2ϩ transient ponential functions (Fig. 3A). We found that there is a decaying induced by 5 mM caffeine or DMPX is equivalent to the summation distribution for spark amplitude (Fig. 3B) and a bell-shaped distri- of 1,582 or 1,701 subsurface sparks (Fig. 4D), respectively.

Ouyang et al. PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12261 Downloaded by guest on September 24, 2021 Fig. 4. Ca2ϩ sparks and secretion in DRG neurons. (A and C)Ca2ϩ sparks as the building blocks of caffeine- and DMPX-induced global Ca2ϩ transients. Curve scan imaging was performed as in Fig. 2. DMPX (A) and caffeine (C)at5mM elicited sparks quickly fused into globe Ca2ϩ transients. An expanded vertical scale was used in C to highlight the leading sparks, rendering the flattened top Fig. 5. Spark-secretion coupling. (A) Spark response to 1 mM caffeine. Time courses of solitary or bursting sparks at marked sites are shown beneath the because data are out of scale. (B) ⌬Cm responses to 5 mM DMPX. Arrow denotes the application of DMPX. (D) Relationship between peak Ca2ϩ tran- image. (B) Simultaneously recorded Cm (line plot) and spark production (bar graph) in 1 mM caffeine. C traces of 3-s length from 41 cells were averaged sients (⌬F͞F0) and ⌬Cm (measured 3 s after caffeine or DMPX application). The m 2 and shown on the top, with control traces obtained from the same cells before equivalent number of subsurface sparks N ϭ (⌬F͞F0,CT ϫ 4␲r )͞(⌬F͞F0,sk ϫ 2 2ϩ caffeine application. Linear fitting to the average C trace in caffeine yielded ␲W ͞4), where ⌬F͞F0,CT refers to the peak Ca transient, r (ϭ10 ␮m) is the m a slope factor of 4.9 fF⅐sϪ1.(C)C changes associated with individual sparks. radius of a typical cell, and ⌬F͞F0,sk (ϭ0.32) and W (ϭ2.05 ␮m) are the peak and m the full width at half maximum of a spark, respectively. Filled diamonds, (Top) Total of 897 traces with a spark event starting at t ϭ 400 ms were aligned thapsigargin pretreatment (10 ␮M 20 min), 5 mM caffeine; open diamonds, 5 and averaged by using the spark onset as the guide. The trailing plateau of the mM caffeine; open circles, 5 mM DMPX. n ϭ 5–14 for each data point. averaged trace was due to bursting spark activity (A and Figs. 1 and 2). (Middle) To reveal specific Cm signal linked to the average spark, the linear rising Cm component was estimated from the data 0–400 ms before the sparks and was then subtracted from the averaged Cm trace. Dashed lines mark the Fig. 4B shows typical time course of Cm change measured in cells 2ϩ levels 0–400 ms before and 100–500 ms after the onset of the average spark, free of Ca indicator under otherwise identical conditions. On ⌬ Ͻ Ͻ ⌬ Ϯ ϭ respectively. (Bottom) Statistics of Cm at 100-ms intervals. **, P 0.01; *, P average, we detected a Cm of 69.9 7.5fF(n 14) at 5 mM 0.05 vs. control (0–400 ms before the spark). caffeine, and 107.7 Ϯ 20.0fF(n ϭ 6) at 5 mM DMPX (Fig. 4D), which corresponds to the secretion of Ϸ140 and 215 vesicles ϭ (assuming 1 fF 2 vesicles) (11), respectively. Inhibiting the ER average results of 897 spark–Cm pairs from 41 cells are shown in Fig. 2ϩ 2ϩ Ca pump by thapsigargin virtually abolished the Ca and ⌬ Cm 5C. Because of the frequent bursting spark activity, the average responses to 5 mM caffeine (Fig. 4D). Thus, we conclude that spark displayed an early peak followed by a sustained plateau (Fig. ϩ spark-mediated store Ca2 release stimulates vesicle secretion 5C Top); its signal integral (0–500 ms from the onset) was equiv- ϩ independently of membrane excitation and Ca2 influx. Given the alent to those of 2.2 solitary sparks. Importantly, we uncovered a above estimates of the equivalent number of sparks (see above), we small but significant Cm increase associated with the average spark further inferred the spark-secretion coupling probability to be one (0.14 Ϯ 0.03 fF between 100–500 ms after the onset of the spark; vesicle per 11.3 or 7.9 sparks in response to 5 mM caffeine or P Ͻ 0.01 vs. 0.0 Ϯ 0.03 fF between 0–400 ms before the spark; Fig. DMPX, respectively. 5C Middle). As a control, we detected no significant changes on The spark-secretion coupling probability was further determined randomly chosen Cm traces (i.e., not aligned to specific spark on the basis of the Cm rising slope and the rate of spark occurrence events) in 1 mM caffeine (n ϭ 812, data not shown). These in indicator-loaded cells exposed to 1 mM caffeine (Fig. 5A). Under measurements not only substantiate the notion on spark-mediated Ϫ1 these experimental conditions, the rising rate of Cm was 4.9 fF⅐s vesicle release, but also suggest the coupling probability to be (data from 41 cells, Fig. 5B Upper), i.e., Ϸ9.8 vesicles per s in a cell. approximately one vesicle per 7.9 sparks. It is noteworthy that a Concomitantly, the spark rate of occurrence was 10.3 (100 significant increase in Cm was first detected with a latency of Ϸ200 ␮m)Ϫ1⅐sϪ1 (data from 41 cells) as seen in the curve scan images (Fig. ms (Fig. 5C Bottom), suggesting that the spark-mediated vesicle 5B Lower). Assuming that curve scan imaging registers sparks fusion is of slow kinetics or involves multiple intermediate steps. within a 2-␮m zone centered at the line, the spark rate of occurrence inacellof20␮m diameter should be 65 sparks per s on the entire Store Ca2؉ Release Modulates Excitation–Secretion Coupling. Neural subsurface shell. These numbers led to an estimate of the coupling secretion is a superlinear function of Ca2ϩ concentration (30, 31), probability to be one vesicle per 6.6 sparks in 1 mM caffeine. implicating a possible synergism between sparks and Ca2ϩ entry in Next, we sought to resolve whether any Cm changes coincided controlling exocytosis. To appraise this possibility, we measured with individual sparks. For this, confocal imaging and Cm recording depolarization-induced vesicle secretion in the absence and pres- were precisely synchronized by marking the image with a 1-ms LED ence of a RyR antagonist (whole-cell dialysis of 50 ␮M ryanodine). flash lit at the onset of Cm recording. We recorded a large quantity Under whole-cell patch-clamp conditions, 200-ms depolarization to of sparks with the corresponding Cm noise below a selection 0 mV induced 460 Ϯ 200 fF ⌬Cm (n ϭ 6, Fig. 6A Left). After 11-min criterion (Ͻ35 fF at 1.24-ms resolution). They were then aligned recording without ryanodine dialyzed into the cell, same depolar- and signal-averaged by using the onset of solitary sparks or the onset ization induced a ⌬Cm that was reduced by 50% (due to run down) of the first sparks in bursting events (Fig. 5A) as the guide. The (Fig. 6A Left). By contrast, such depolarization-induced ⌬Cm was

12262 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408494102 Ouyang et al. Downloaded by guest on September 24, 2021 caused a significantly elevated CGRP release and potentiated the CGRP release in response to endotoxin. Conversely, inhibition of RyRs or ER Ca2ϩ-ATPase abolished the LPS-induced CGRP secretion, whereas selective inhibition of IP3Rs with xestospongin C (12 ␮M) exerted no significant effect. These findings suggest that RyR-mediated release is required for endotoxin-induced neuropeptide secretion. Discussion Neural Ca2؉ Sparks. In the present study, we have provided an unequivocal demonstration of sparks as the elemental store Ca2ϩ release events in a mammalian sensory neuron. Arising from RyR3 mostly on SSC, these sparks occur spontaneously at a low frequency in resting DRG cells, but can be readily activated with the RyR sensitizers, caffeine and DMPX. During excitation, spark production is triggered by physiological Ca2ϩ influx via the CICR mechanism such that the evoked sparks amplify the trigger Ca2ϩ entry, allowing for synergistic interaction between Ca2ϩ release and Ca2ϩ entry in the regulation of local Ca2ϩ signaling events such as exocytosis. The possibility for the existence of sparks in neurons was first suggested on the basis of excessive ‘‘noise’’ or bright pixels revealed by histogram analysis of neuronal Ca2ϩ images in which no dis- cernible local events could be resolved (32). Meanwhile, Koizumi et al. (33) showed that, in nerve growth factor-differentiated PC12 cells or cultured hippocampal neurons, the elementary Ca2ϩ release events arise from both RyR and IP3R, and exhibit 2-fold greater spatial width and at least 20-fold longer duration than a typical cardiac spark. Later, Llano et al. (7) visualized RyR- mediated spontaneous Ca2ϩ release at presynaptic terminals, where individual events last for a few seconds and spread by 5–10 ␮m along the processes. These local Ca2ϩ release events along neural process might involve CICR among multiple release sites and 2ϩ 2ϩ 2ϩ therefore reflect compound Ca sparks. Most recently, local Ca Fig. 6. Ca sparks contribute to depolarization- and endotoxin-induced release events called Ca2ϩ syntillas have been characterized at exocytosis and neuropeptide secretion. (A) Intracellular dialysis of ryanodine 2ϩ hypothalamic neural terminals with a nonconfocal technique (34). (Ry, 50 ␮M) caused smaller [Ca ]i transients and greatly reduced ⌬Cm during Ϫ ⌬ Mechanistically, hypothalamic syntillas can be activated by depo- depolarization (200 ms, 80 mV to 0 mV). Cm was measured at 2 and 11 min ϩ ϩ 2ϩ 2 2 after whole-cell dialysis with or without ryanodine. [Ca ]i transients larization in a Ca -independent manner, whereas Ca influx is 2ϩ (⌬[Ca ]i) were expressed as the percent of that at 2 min. (B) Statistical analysis. obligatory to depolarization-evoked sparks in DRG neurons. Taken ϩ Data were expressed as the percent of the control values at the same time together, the recording of Ca2 syntillas and sparks signifies the 2ϩ ⌬ 2ϩ point. Ryanodine dialysis for 5 or 11 min decreased Ca transients and Cm, beginning of investigation of store-mediated Ca signaling in ϭ Ͻ Ͻ but not the ICa. n 6 for each group; **, P 0.01; *, P 0.05 vs. the neurons on the smallest physiological scale. corresponding control. (C) CGRP release in cultured neonatal DRG neurons. Compared to prototypical sparks in muscles (⌬F͞F Ϸ 0.7, full Ϯ ⅐ Ϫ1 0 The basal level of CGRP-like immunoactivity (CGRP-LI) was 48.2 6.7 pg ml . width at half maximum Ϸ 2.0 ␮m, release duration of 5–10 ms), Caffeine (Caf, 3 mM) potentiated LPS-stimulated (1 ␮g⅐mlϪ1) CGRP release, ␮ ␮ DRG sparks are characterized by a halved amplitude (0.3 whereas thapsigargin (TG, 10 M) and ryanodine (Ry, 10 M), but not xesto- ⌬ ͞ spongin C (XeC, 12 ␮M), suppressed the LPS-induced CGRP release. n ϭ 5–8 F F0), similar spatial extension, but markedly prolonged re- Ϸ independent experiments. **, P Ͻ 0.01; *, P Ͻ 0.05 vs. control (ctrl); &, P Ͻ 0.05 lease duration ( 40 ms), displaying a monoexponential distri- vs. LPS alone. bution. The small amplitude combined with the prolonged release duration predicts that Ca2ϩ release flux underlying a spark must be smaller in DRG neurons than muscle cells. The reduced by Ϸ90% of the initial value in the presence of intracellular bursting behavior as well as the long and exponentially distrib- 50 ␮M ryanodine (Fig. 6A Right). Compared to control values at 11 uted release duration make DRG sparks most distinctive, be- ϩ min, ryanodine caused a 44% reduction in Ca2 transient amplitude cause in striated muscles, tight regulation endows sparks with (P Ͻ 0.01) without affecting ICa (Fig. 6B), and inhibited vesicular brief (5–10 ms) and preferred release duration (19, 35) and secretion by 60% (P Ͻ 0.01) because of the nonlinear relation strong use-dependent inactivation (36, 37). The unique proper- 2ϩ between [Ca ]i and secretion. These results indicate that spark- ties of DRG spark termination and refractoriness might stem mediated release acts in synergy with Ca2ϩ influx in the modulation from the fact that DRG RyR3 in planar lipid bilayers does not of excitation–secretion coupling. inactivate even in the presence of 10 mM Ca2ϩ on the cytosolic side (25). PHYSIOLOGY Inhibition of Sparks Prevents Endotoxin-Induced CGRP Release. Se- cretion in DRG cells not only is linked to membrane electrical Spark-Secretion Coupling. The vast majority of DRG sparks are activities, but also is modulated by receptor-mediated signal trans- found in 1-␮m outmost shell of the cytoplasm, as is the case with duction. For instance, we have recently shown that lipopolysaccha- RyR3 immunoreactive aggregates. As such, RyR3 sparks are well ride (LPS), known also as endotoxin, stimulates the release of positioned to mediate reciprocal communication between the CGRP, a 37-aa pain-related neuropeptide, from neonatal rat DRG plasma membrane and the intracellular Ca2ϩ store. In this regard, neurons in culture (23). Using a RIA, we examined the possible we have demonstrated that sparks are directly triggered by local involvement of CICR production in the endotoxin-induced CGRP physiological Ca2ϩ entry, and conversely, subsurface sparks are secretion (Fig. 6C). We found that incubation with 3 mM caffeine equally well poised to mediate the retrograde signaling, e.g., Ca2ϩ-

Ouyang et al. PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12263 Downloaded by guest on September 24, 2021 dependent vesicular secretion. Multiple lines of evidence have been is in general agreement with the observation that CICR contributes presented to show role of sparks in triggering and modulating to asynchronous exocytosis at frog motor nerve terminals (39). vesicular secretion from somata of DRG neurons. First, activation Moreover, it has also been shown that RyR-mediated Ca2ϩ release of sparks by caffeine or DMPX suffices to trigger significant vesicle in presynaptic terminals of basket cells underlie spontaneous, release indexed by Cm increase. This process requires neither multivesicular secretion, causing large-amplitude miniature inhib- membrane excitation nor Ca2ϩ influx; yet, suppression of spark itory post synaptic currents in Purkinje cells (7). In hippocampal production by pretreatment with thapsigargin abolishes the caffeine neurons, CICR has also been shown to modulate synaptic trans- effects. Second, by signal averaging of sparks and the corresponding mission because excitatory postsynaptic potentials are smaller in the Cm traces, we were able to resolve a miniscule Cm changes tempo- presence of ryanodine (9). Collectively, the present and previous ϩ rally linked to individual solitary or bursting sparks (Fig. 5C). findings unveil a role for store Ca2 release in secretion functions Furthermore, we found that CICR amplifies and works in concert at the presynaptic terminals and cell body of neurons. The present with the Ca2ϩ entry in modulating excitation–secretion coupling. finding also sheds light on the long-sought physiological significance Inhibition of CICR suppresses the major component (Ϸ60%) of of neuronal SSC that forms nanoscopic junctions with the plasma Ca2ϩ-dependent secretion and abolishes the endotoxin-mediated membrane. release of CGRP. In summary, we have demonstrated that sparks arising from ϩ In theory, individual vesicle exocytosis might be coupled to RyR3 on SSC underlie excitation-Ca2 release coupling in a solitary or bursting sparks in a stochastic manner. Indeed, triggering mammalian sensory neuron. Owing to their subsurface localization, sparks alone can directly trigger vesicle secretion. When acting in exocytosis by sparks appears to be a probabilistic, rather than ϩ deterministic, process. Three different methods were applied to synergy with the surface Ca2 influx, subsurface sparks play an estimate the coupling probability based on the relationship between important role in the modulation of excitation–secretion coupling. peak ⌬F͞F0 and ⌬Cm in response to 5 mM caffeine or DMPX, the That RyR inhibition suppresses endotoxin-induced secretion of an relationship between the spark rate and the rising slope of Cm excitatory pain-related peptide implicates RyR3 of DRG as a novel during 1 mM caffeine, and the relationship between averaged target for analgesic therapeutics. These findings, as well as the approaches presented here, may have general implications to individual sparks and the corresponding Cm changes. These esti- 2ϩ mates fell within a range between one vesicle per 6.6 sparks to one understanding store-mediated Ca signaling in neuronal activities vesicle per 11.3 sparks. This low spark–secretion coupling efficiency (transmitter release, somatic secretion, excitability, synaptic plas- is in general agreement with the finding in the CNS synapse calyx ticity, etc.) at the elementary release level. of Held, where 10 or more Ca2ϩ channels control the release of a single vesicle (38). The rather long latency (Ϸ200 ms) from the We thank Dr. Clara Franzini-Armstrong for suggestion on ganglion onset of the average spark to the first detection of a significant C staining; Drs. Zuoping Xie, Lu-Yang Wang, and Shi-qiang Wang for m critical comments; and Dr. Qian Hu for technical support. This work was increase, as shown in Fig. 5C, further suggests that multiple steps or supported by Major State Basic Research Development Program (X.W., slow kinetics underlie the spark-mediated vesicle fusion and Z.Z., and H.C.), National Natural Science Funds of China (D.Y., Z.Z., secretion. and H.C.) and the National Institutes of Health intramural research The demonstration of spark-secretion coupling in DRG neurons program (H.C.).

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